Polycationic amphiphiles as antimicrobial agents

ABSTRACT

The present disclosure provides an antimicrobial composition including a polycationic amphiphile compound, and the method of making and the method of using such a compound or composition. The compound having the formula 
     
       
         
         
             
             
         
       
     
     R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 10 , or R 11  is H or a C 1-12  alkyl unsubstituted or optionally substituted with a functional group such as —OH, —OR′, —NH 2 , —NHR′, —N—C(O)R′, —N—C(O)CR′═CR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF 3 , —OCF 3 , halogen, benzyl, o-vinylbenzyl, m-vinylbenzyl, p-vinylbenzyl, phenyl, allyl, and substituted allyl. R 7 , R 8  or R 9  is a C 1-12  alkyl unsubstituted or optionally substituted with a functional group such as —OH, —OR′, —NH 2 , —NHR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF 3 , and —OCF 3 . R′ is H or a C 1-4  alkyl. X or Y is a halogen. m and n are integers in the range from 1 to 25.

PRIORITY CLAIM AND CROSS-REFERENCE

This application is a continuation-in-part of U.S. application Ser. No. 15/034,375, filed May 4, 2016, which is a national phase application of International Application No. PCT/US2014/064122, filed Nov. 5, 2014, which claims the benefit of U.S. Provisional Application No. 61/900,037, filed Nov. 5, 2013; U.S. Provisional Application No. 62/039,265, filed Aug. 19, 2014; and U.S. Provisional Application No. 62/059,216, filed Oct. 3, 2014, which applications are expressly incorporated by reference herein in their entireties.

FIELD

The disclosure relates to antimicrobial compositions and related methods. More particularly, the disclosed subject matter relates to a composition comprising a polycationic amphiphile, the method of making and the method of using such an amphiphile for antimicrobial use.

BACKGROUND

The preparation of chemical agents to counter the spread of human pathogens has been a challenge long before the term medicinal chemistry was coined. From the fermentation of beverages to the preparation of bleach, the facile production of compounds to minimize the pathogenic effects of microbes has been a key concern. Development of bacterial resistance to even the most potent antibiotics has ensured that continued research into antimicrobial compounds will remain crucial.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure are best understood from the following detailed description when read with the accompanying figures. It is noted that, in accordance with the standard practice in the industry, various features are not drawn to scale.

FIG. 1 illustrates the life cycle of a biofilm and the inspirations for developing polycationic amphiphiles to inhibit or eradicate biofilms in accordance with some embodiments.

FIG. 2 illustrates the approach of mimicking antimicrobial peptides using exemplary structures (Chimera-rendered space filling models of lowest energy conformations) showing the cationic regions (light regions) and hydrophobic regions (dark regions).

FIG. 3 shows images of crystal violet staining and confocal microscopy. Left: Submerged S. aureus biofilms 16 h post-treatment with the indicated final well concentration of compound. Middle: Biofilms stained with 1% crystal violet. Right: Confocal microscopy images of biofilms 16 h post-treatment.

FIG. 4. Structures of quaternary ammonium compounds (QACs) of Tables 4 and 5. (A) MonoQACs found in commercial disinfectants, (B) monoQACs, (C) bisQACs, (D) a tetraQAC, (E) trisQACs, and (F) paraquats (PQ), metaquats (MQ), and parametaquat (PMQ). Compound abbreviations such as 12,3,0 indicate alkylation on nitrogen leading to quaternization, either as number of carbons in alkyl chain (12=dodecyl chain) or other substituent. Number 0 indicates no substitution, thus a tertiary amine. Linker lengths appear in parentheses. Bn=benzyl, Pyr=pyridinium, 3A=allyl, Bn-8=CH₂-Ph-4-(C₈H₁₇), 11-SH=C₁₁H₂₂SH. All counterions are bromide except for structures compounds (Bn,12); (10,10); and (Pyr, 16) (chloride), as well as compounds (12,2,1,2,12) and (12,3,1,3,12) (bis-bromide mono-iodide).

SUMMARY OF THE INVENTION

This Summary is provided to present a summary of the invention to briefly indicate the nature and substance of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

The present disclosure provides an antimicrobial composition comprising a compound which is a polycationic amphiphile such as a triscationic or tetracationic amphiphile, the method of making such an antimicrobial composition, and the method of using such a compound or composition for antimicrobial use. The compound or the composition provided in the disclosure has an ability to kill or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, to attenuate the severity of a microbial infection, or to inhibit formation of a biofilm or eradicate pre-established biofilms (i.e. antibiofilm use).

Some embodiments of the present disclosure provide an antimicrobial composition comprising a compound having the formula

wherein:

R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is H or a C₁₋₁₂ alkyl unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —N—C(O)R′, —N—C(O)CR′═CR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, —OCF₃, halogen, benzyl, o-vinylbenzyl, m-vinylbenzyl, p-vinylbenzyl, phenyl, allyl, and substituted allyl;

R₇, R₈ or R₉ is a C₁₋₁₂ alkyl unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′ —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃,

R′ is H or a C₁₋₄ alkyl;

X or Y is a halogen (in the form of anion); and

m and n are integers in the range from 1 to 25 (e.g., in the range from 10 to 16).

X or Y can be any halogen including but not limited to fluorine, chlorine, bromine, iodine, and any combination thereof. In some embodiments, R₇, R₈ or R₉ as the linkers between two nitrogen atoms can be a C₂₋₅ alkyl unsubstituted or optionally substituted. For example, linkers R₇, R₈ or R₉ can be —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, or —CH₂CH₂CH₂CH₂CH₂—. In some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ may be a C₁₋₄ alkyl unsubstituted or optionally substituted. In some compounds, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is a C₁₋₁₂ alkyl substituted with a functional group selected from the group consisting of —SH, allyl, and substituted allyl.

In some embodiments, ionization of the middle nitrogen are optional. For example, ionization of the middle nitrogen (N) atom with Y and substitution of either R₃ or R₄ in Formula (I) may be optional. Ionization of the two middle nitrogen (N) atoms with Y and substitution of either R₃ or R₄ and either R5 or R6 in Formula (II) may be also optional.

In another aspect, the present disclosure provides a method of making the antimicrobial composition comprising a compound having the formula (I) or (II) as described. The method comprises mixing an effective amount of a compound having the formula (I) or (II) and a carrier.

The present disclosure also provides a method of killing or inhibiting microbial growth, comprising applying the antimicrobial composition comprising a compound having the formula (I) or (II) as described. The antimicrobial composition or the compound is used to kill or inhibit growth of at least one group of microorganisms selected from the group consisting of bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm, or disperse or eradicate a pre-established biofilm.

In another aspect, the present disclosure further provides a film or coating comprising a compound

The compound having the formula (I) or (II) is grafted onto a solid surface. As described, R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ can be H or a C₁₋₁₂ alkyl (e.g., a C₂₋₅ alkyl) unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —N—C(O)R′, —N—C(O)CR′═CR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, —OCF₃, halogen, benzyl, o-vinylbenzyl, m-vinylbenzyl, p-vinylbenzyl, phenyl, allyl, and substituted allyl. R₇, R₈ or R₉ can be a C₁₋₁₂ alkyl (e.g., a C₂₋₅ alkyl) unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃. R′ is H or a C₁₋₄ alkyl. X or Y is a halogen, including fluorine, chlorine, bromine, iodine and any combinations thereof.

m and n are integers in the range from 1 to 25 (e.g., in the range from 10 to 16).

For example, the compound grafted on a solid surface may have a structure as shown by the formula

L is a linker comprising a functional group. In some embodiments, R4 is a methylene group optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃. In some embodiments, in such a film or coating comprising a compound having the formula (I) or (II), at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is a C₁₋₁₂ alkyl substituted with a functional group (e.g., —SH, allyl, and substituted allyl).

The film or coating is configured to kill or inhibit growth of at least one group of microorganisms selected from the group consisting of bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm or eradicate pre-established biofilms.

DETAILED DESCRIPTION

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.

DEFINITIONS

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

The term “antimicrobial” refers to an ability to kill or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, or to attenuate the severity of a microbial infection. The antimicrobial compounds or compositions of the present invention are compounds or compositions that may be used for cleaning or sterilization, or may be used in the treatment of disease and infection. The applications may include both in vitro and in vivo antimicrobial uses. “Applying” an antimicrobial composition may include administrating a composition into a human or animal subject.

The term “biofilm” as used herein refer to a film formed by a group of microorganisms adhered together. The term “antibiofilm” as used herein refer to an ability to kill, disperse and/or eradicate a pre-established biofilm.

The term “alkyl” as used herein refers to a straight chain, cyclic, branched or unbranched saturated or unsaturated hydrocarbon chain containing 1-25 carbon atoms, such as methyl, ethyl, propyl, tert-butyl, n-hexyl and the like. “A C₁₋₁₂ alkyl” as used herein refers to an alkyl group having a number of carbon atoms selected from 1 to 12.

The term “optionally substituted” means that group in question may be unsubstituted or it may be substituted one or several times, such as 1 to 3 times or 1 to 5 times. For example, an alkyl group that is “optionally substituted” with 1 to 5 chloro atoms, may be unsubstituted, or it may contain 1, 2, 3, 4, or 5 chlorine atoms. Substituted chemical moieties include one or more substituents that replace hydrogen.

The term “minimum inhibitory concentration (MIC)” means the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. MIC values against bacteria, for example, the Gram-positive Staphylococcus aureus and Enterococcus faecalis and the Gram-negative Escherichia coli and Pseudomonas aeruginosa were determined by standard methods. See also P. A. Wayne, Methods for Dilution Antimicrobial Tests for Bacteria that Grow Aerobically; Approved Standard, Ninth Edition, 2012, CLSI Document M07-A9, Vol. 32 No. 2.

The term “the minimum biofilm eradication concentration (MBEC)” of a compound is defined as the lowest concentration of compound dosed against a previously established bacterial biofilm that leads to a clear well (optical density of less than 0.1) when the treated biofilm is regrown in fresh media, indicating >95% clearance of bacteria. A regrowth assay was used to establish the MBEC of a compound to evaluate the antibiofilm activity. See also H. Ceri, M. Olson, D. Morck, D. Storey, R. Read, A. Buret, B. Olson, Methods Enzymol. 2001, 337, 377.

The present disclosure provides an antimicrobial composition comprising a compound which is a polycationic (e.g., triscationic, tetracationic or the like) amphiphile, and the method of making such an antimicrobial compound or composition, and the method of using such a compound or composition for antimicrobial use. The compound or the composition provided in the disclosure has an ability to kill or inhibit the growth of microorganisms, including but not limited to bacteria, viruses, yeast, fungi, and protozoa, or to attenuate the severity of a microbial infection. The compound or the composition provided in the disclosure has an ability to inhibit or eradicate pre-established biofilms (i.e. antibiofilm use) formed by the microorganisms.

Biofilms are complex communities of bacteria that exist in a self-produced matrix of polysaccharides, proteins, and extracellular DNA. According to the US Centers for Disease Control and Prevention, biofilm infections are responsible for over 65% of nosocomial and foreign-device infections. Bacterial cells exhibiting the biofilm phenotype are 100-1000 times more resistant to standard antibiotics, as traditional molecular targets can be latent in the biofilm state and the presence of the extrapolymeric substance (EPS) interferes with antibiotic localization. As illustrated in FIG. 1, the biofilm life cycle consists of four stages: (1) Adhesion—planktonic cells undergo a phenotypic change and aggregate on a surface; (2) EPS production—secretion of polysaccharides, extracellular DNA, and proteins; (3) Maturation—development of biofilm architecture; and (4) Dispersion—phenotypic reversion to planktonic cells. Bacteria utilize many chemical signals to orchestrate this complex lifestyle, and significant efforts have been made to mimic these chemical classes to inhibit biofilm formation; however, synthetic compounds that eradicate pre-established biofilms have been sparsely reported.

Amphiphiles, molecules with both polar and non-polar regions, have been a mainstay of antimicrobial compounds. The use of soap, with ready availability and a simple anionic amphiphilic structure, has developed into one of the greatest advances in human health. The use of benzalkonium chloride, with a comparably straightforward cationic amphiphilic structure, propelled this revolution into the 20^(th) century, where a number of amphiphilic compositions such as LYSOL® brand products have become commonplace in the household.

Because a variety of amphiphiles share a common method of action, namely membrane disruption, both anionic and cationic versions have been the focus of antimicrobial development campaigns in recent decades. Cationic amphiphiles have been regarded as membrane disruptors, capitalizing on electrostatic interactions with the predominantly anionic bacterial cell membrane, followed by intercalation of the non-polar chain, which leads to membrane disruption and ultimately bacterial cell lysis. Multiply charged amphiphilic species having polyanionic structures have been developed, perhaps taking inspiration from nature's antimicrobial peptides. For example, a series of dendritic amphiphiles having three carboxylate groups allow for attachment to surfaces (Scheme 1, left).

The inventors have developed cationic amphiphilic structures and have correlated the structures to antimicrobial activity. The inventors have developed quaternary ammonium amphiphiles based on an all-carbon aromatic core (A), an inexpensive TMEDA (N,N,N′,N′-tetramethyl ethylenediamine) core (B), as well as bis-pyridinium structures of both 4,4′ (C) and other geometries (D), as illustrated in Scheme 1. The inventors have further developed a series of novel polycationic (e.g., triscationic, tetracationic or the like) amphiphile compounds for antimicrobial uses in the present disclosure. These compounds can be used in an antimicrobial composition, or used as a film or coating after grated onto a solid surface.

In one aspect, the present disclosure provides an antimicrobial composition comprising a compound having the formula

wherein:

R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is H or a C₁₋₁₂ alkyl unsubstituted or optionally substituted with a functional group such as —OH, —OR′, —NH₂, —NHR′, —N—C(O)R′, —N—C(O)CR′═CR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, —OCF₃, halogen, benzyl, o-vinylbenzyl, m-vinylbenzyl, p-vinylbenzyl, phenyl, allyl, and substituted allyl;

R₇, R₈ or R₉ is a C₁₋₁₂ alkyl unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃;

R′ is H or a C₁₋₄ alkyl or optionally substituted;

X or Y is a halogen (in the form of anion); and

m and n are integers in the range from 1 to 25.

For example, in some embodiments, R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ can be H or a C₁₋₄ alkyl unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃. In some embodiments, at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is a C₁₋₁₂ alkyl substituted with a functional group such as —SH, allyl, substituted allyl or the like. X or Y can be any halogen in the form of ion including but not limited to fluorine, chlorine, bromine, iodine, and any combination thereof. X or Y can be also tosylate, citrate, any suitable anions or combinations thereof. In some embodiments, X or Y is chlorine or bromine (i.e. chloride or bromide). m and n can be integers in the range from 5 to 25 (e.g., in the range from 10 to 16). m can be equal or not equal to n. For example, in some embodiments, at least one of m and n is 12.

R₇, R₈ or R₉ as the linkers between two nitrogen atoms can be a C₂₋₅ alkyl unsubstituted or optionally substituted. R₇, R₈ or R₉ may be a repeating methylene group unsubstituted in the form of —(CH₂)_(s)—, or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃, s is an integer, for example, in the range from 1 to 12. The value of s in R₇, R₈ or R₉ can be different from each other.

In some embodiments, linkers R₇, R₈ or R₉ is —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, or —CH₂CH₂CH₂CH₂CH₂—. For example, the compound having the formula (I) can have the formula

In the compound having the formula (I-2), (I-3), (I-4) or (I-5) (e.g., I-3), at least one of R₁ and R₂, and at least one of R₃ and R₄, and at least one of R₅ and R₆, can be methyl in some embodiments. In some of these compounds, m and n are 12, one of R₃ and R₄ is selected from a group consisting of ethyl, propyl and allyl, and the other groups among R₁, R₂, R₃, R₄, R₅, and R₆ are methyl.

Some exemplary compounds described in the present disclosure are denoted using a combination of numbers. For example, in some compounds having the formula (I-2), (I-3), (I-4) or (I-5), both R₁ and R₂, both R₅ and R₆ are methyl. At least one of R₃ and R₄ is methyl. Assuming R₃ is methyl, R₄ is optionally H or an alkyl having x carbons, which is substituted or optionally substituted. Such a compound is denoted as compound [m, s, x, s, n], compound (m, s, x, s, n), or compound m, s, x, s, n. x is the number of carbon atoms in R₄. When R₄ is H, x is zero (labelled as 0′ for a central nitrogen atom which is ionized and also substituted with R₄ being H). When R₄ does not exist and the central nitrogen atom is not ionized, x is labelled as 0. For example, such a compound having the formula (I-3), in which R₃ is methyl, R₄ is ethyl and both m and n are 12, is denoted as compound [12, 3, 2, 3, 12]. When R₄ is an allyl group, x is denoted as “3A.” For another example, such a compound having the formula (I-3), in which R₃ is methyl, R₄ is allyl and both m and n are 12, is denoted as compound [12, 3, 3A, 3, 12].

Similarly, the compound having the formula (II) can have the formula

In the compound having the formula (II-2), (II-3), (II-4) or (II-5), at least one of R₁ and R₂, and at least one of R₃ and R₄, at least one of R₅ and R₆, and at least one of R₁₀ and R₁₁ can be methyl in some embodiments. In some of these compounds, for example, having the formula (II-2), m and n are 12, one of R₃ and R₄ is selected from a group consisting of ethyl, propyl and allyl, and the other groups among R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, and R₁₁ are methyl.

Some exemplary compounds described in the present disclosure are denoted using a combination of numbers. For example, in some compounds having the formula (II-2), both R₁ and R₂, and both R₁₀ and R₁₁ are methyl. At least one of R₃ and R₄ is methyl. At least one of R₅ and R₆ is methyl. Assuming R₃ and R₅ are methyl, R₄ and R₆ are H or an alkyl having x₄ and x₆ carbons, respectively, which is substituted or optionally substituted. Such a compound is denoted as compound [N4-m, x₄, x₆, n], compound (N4-m, x₄, x₆, n), or compound N4-m, x₄, x₆, n. When R₄ or R₆ is H, x₄ or x₆ is zero (labelled as 0′ for a central nitrogen atom which is ionized and also substituted with R₄ or R₆ being H). When R₄ or R₆ does not exist and the central nitrogen atom is not ionized, x₄ or x₆ is labelled as 0. For example, such a compound having the formula (II-2), in which R₃ and R₅ are methyl, R₄ and R₆ are methyl, and both m and n are 12, is denoted as compound [N4-12, 1, 1, 12]. When R₄ and R₆ are an allyl group, x₄ or x₆ are denoted as “3A.” For example, such a compound having the formula (II-2), in which R₃ and R₅ are methyl, R₄ and R₆ are allyl, and both m and n are 12, is denoted as compound [N4-12, 3A, 3A, 12].

The present disclosure also provides a method of making a compound having the formula (I) or (II) as described. Synthesis of the compound having the formula (I) or (II) can be achieved using a corresponding polyamine, particularly a polyamine with desirable cost. The polyamine can be grafted with the substitution groups at both ends or in the middle to form the resulting polycationic compound.

In another aspect, the present disclosure provides a method of making the antimicrobial composition comprising a compound having the formula (I) or (II) as described. The method comprises mixing an effective amount of a compound having the formula (I) or (II) and a carrier such as a solvent, a carrier, an additive, any other suitable ingredient, or combinations thereof.

In some embodiments, the present disclosure also provides an antimicrobial composition comprising a compound having the formula (I) or (II) as described, and a carrier such as a solvent. The antimicrobial composition can also comprise other ingredients and additives. The content of the compound having the formula (I) or (II) can be in any suitable concentration. For example, in some embodiments, such a concentration can be in the range from 0.01 μM to 100 μM, for example, from 0.1 μM to 10 μM. In some embodiments, the content of the compound having the formula (I) or (II) may be at a concentration of from 0.1 wt. % to 5 wt. %, for example, in the range of from 0.2 wt. % to 2.5 wt. %. Examples of the carrier include (or exclude) but are not limited to a solvent. Examples of other additives include (or exclude) but are not limited to surfactants, anti-foaming agents, anti-freezing agents, gelling agents, and combinations thereof. The antimicrobial composition may also comprise a pharmaceutically acceptable carrier or excipient. A pharmaceutically acceptable carrier or excipient suitable for a solid preparation such as tablets or capsules can be, for example, binders (e.g., acacia, gelatin, dextrin, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone), solvents, dispersion media, diluents (e.g., lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate, crystalline cellulose), lubricants (e.g., magnesium stearate, calcium stearate, stearic acid, talc, anhydrous silicic acid), disintegrants (e.g., corn starch, potato starch, carboxymethylcellulose, carboxymethylcellulose calcium, alginic acid), and wetting agents (e.g., sodium laurylsulfate). A pharmaceutically acceptable carrier or excipient suitable for a liquid preparation, such as solutions or suspensions, can be, for example, aqueous vehicles (e.g., water), suspending agents (e.g., acacia, gelatin, methyl cellulose, carboxymethylcellulose sodium, hydroxymethyl-cellulose, aluminum stearate gel), surfactants (e.g., lecithin, sorbitan monooleate, glycerin monostearate), and non-aqueous vehicles (e.g., glycerin, propylene glycol, vegetable oil). Moreover, liquid preparations may contain preservatives (e.g., p-hydroxybenzoic acid methyl ester, p-hydroxybenzoic acid propyl ester), flavors, and/or coloring agents. The antimicrobial composition in this disclosure can be formulated to be in any suitable form, including but not limited to liquid, gel and paste.

The present disclosure also provides a method of killing or inhibiting microbial growth, comprising applying the antimicrobial composition comprising a compound having the formula (I) or (II) as described. The antimicrobial composition or the compound is used to kill or inhibit growth of at least one group of microorganisms selected from the group consisting of bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm or eradicate a pre-established biofilm. Examples of a suitable method include but are not limited to pouring, spraying, any other suitable methods and any combinations thereof.

In another aspect, the present disclosure further provides a film or coating comprising a compound

as described.

The compound having the formula (I) or (II) is grafted onto a solid surface. As described, R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ can be H or a C₁₋₁₂ alkyl (e.g., a C₂₋₅ alkyl) unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —N—C(O)R′, —N—C(O)CR′═CR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, —OCF₃, halogen, benzyl, o-vinylbenzyl, m-vinylbenzyl, p-vinylbenzyl, phenyl, allyl, and substituted allyl. R₇, R₈ or R₉ can be a C₁₋₁₂ alkyl (e.g., a C₂₋₅ alkyl) unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃. R′ is H or a C₁₋₄ alkyl. X or Y is a halogen, including fluorine, chlorine, bromine, iodine and any combinations thereof. m and n are integers in the range from 1 to 25 (e.g., in the range from 10 to 16).

For example, the compound grafted on a solid surface may have a structure as shown by the formula

L is a linker comprising a functional group. In some embodiments, R4 is a methylene group optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —NR′₂, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃. In some embodiments, in such a film or coating comprising a compound having the formula (I) or (II), at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is a C₁₋₁₂ alkyl substituted with a functional group selected from the group consisting of —SH, allyl, and substituted allyl. X can be chlorine or bromine. At least one of m and n may be 12.

The film or coating is configured to kill or inhibit growth of at least one group of microorganisms selected from the group consisting of bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm or eradicate pre-established biofilms.

EXAMPLES

A series of polycationic amphiphiles have been prepared. Such polycationic amphiphiles have powerful antimicrobial activities. The examples described below are for the purpose of illustration only.

1. Two Triscationic Amphiphiles for Eradicating Established Biofilm

Cationic amphiphiles have been implemented, both biologically and commercially, to thwart bacterial infections. In the biological arena, both eukaryotic and prokaryotic organisms exploit antimicrobial peptides (AMPs) to prevent and eliminate prokaryotic colonization. AMPs strategically incorporate a variety of features within the peptide scaffold to eradicate infections including cationic, amphipathic, and hydrophobic residues. Representative AMPs with various secondary structures (e.g. linear, alpha helical) with different physical properties (charged or hydrophobic) illustrated in different shades are depicted in FIG. 2A. In FIG. 2, the light regions are positively charged and the black regions are hydrophobic.

In contrast to inhibitors, which prevent the formation of biofilms, and dispersers, which promote the transition to planktonic cells, compounds that completely eradicate established biofilms at practical concentrations (<500 μM) have been sparsely reported. Based on this key deficiency, and inspired by previous work on antimicrobial peptides (AMP) derivatives, the inventors hypothesized that quaternary ammonium cations (QACs) could serve as simplified AMPs and eradicate pre-existing biofilms. To the best knowledge of the inventors, there have been no documented studies of their effects on biofilms.

Scheme 1 shows synthesis and structures of mono- and bis-QACs (structures 1-7′,9′,11′ in this section), paraquat (PQ)-11,11 (structure 8′), and tris-QACs (10′,12′). Tris-QACs (compounds 10′,12′) have been synthesized in accordance with some embodiments in the present disclosure. The QAC structures shown in Scheme 1 are reminiscent of the class of polyamines (e.g., norspermidine), and accordingly the inventors hypothesized that QACs, particularly the polycationic amphiphiles provided in the present disclosure, would possess the potent antibiofilm activity sought. Table 1 shows the minimum inhibitory concentration of the compounds illustrated in Scheme 1. Table 2 shows the minimum biofilm eradication concentration (MBEC) of these compounds.

The inventors' focused library of QACs utilized varied linker length to probe the effect that both charge and three-dimensional structure play in the compounds' biological effect, akin to the structural diversity displayed by AMPs (as shown in FIG. 2B). Recent efforts from the laboratories of the inventors led to the inexpensive preparation of tetramethylethylenediamine (TMEDA)-derived QACs with low micromolar inhibitory activity against both Gram-positive and Gram-negative planktonic bacteria (e.g., compound 12,2,12). In an extension of this TMEDA platform, the compound library was designed to include mono-, bis-, and tris-cations as well as mono-, di-, and triamines with linker lengths of two, three, and five carbons. With the exception of compound 1′ (benzalkonium chloride, a component of many household antiseptics), all synthesized compounds are accessible in one to two steps from commercially available starting materials through simple alkylation chemistry (Scheme 2). The compounds bearing dodecyl side chains demonstrated MIC values in the low micromolar range (Table 1).

TABLE 1 MICs of norspermidine, QACs, and PQ-11,11. MIC (μM) Compound (No.) S. aureus E. faecalis E. coli P. aeruginosa Norspermidine >500 >500 >500 >500 Bn,12 (1′) 8 8 32 63 12,12 (2′) 1 1 8 32 1,2,1 (3′) >500 >500 >500 >500 1,5,1 (4′) >500 >500 >500 >500 12,2,12 (5′) 1 1 2 4 12,3,12 (6′) 2 1 1 4 12,5,12 (7′) 1 1 1 4 PQ-11,11 (8′) 1 1 1 4 12,2,0,2,12 (9′) 1 2 1 4 12,2,1,2,12 (10′) ≦0.25 ≦0.25 0.5 1 12,3,0,3,12 (11′) 1 ≦0.25 ≦0.25 4 12,3,1,3,12 (12′) 0.5 1 1 2

Encouraged by the potency of the QACs against planktonic bacterial cells, the inventors then evaluated the efficacy of compound (12,2,12) against Staphylococcus aureus and Enterococcus faecalis biofilms. Compound (12,2,12) showed significant effects on pre-established biofilms at low micromolar concentrations (Table 2). To both accurately evaluate the antibiofilm activity, a regrowth assay was utilized to establish the minimum biofilm eradication concentration (MBEC) of each compound. Compound (12,2,12) has an MBEC value of 75 μM against both bacteria, approximately two-fold better than the monocationic derivative (12,12) and commercially-utilized compound 1′ (Bn, 12) (Table 2.) It was also found that compounds (1,2,1) and (1,5,1) were unable to eradicate biofilms at the concentrations tested, demonstrating that both the cationic character and alkyl side chains are necessary to retain efficacy.

TABLE 2 MBECs of norspermidne, QACs, and PQ-11,11. MBEC (μM) Compound (No.) S. aureus E. faecalis Norspermidine >200 >200 Bn,12 (1′) 200 200 12,12 (2′) >200 200 1,2,1 (3′) >200 >200 1,5,1 (4′) >200 >200 12,2,12 (5′) 75 ± 25^(a) 75 ± 25^(a) 12,3,12 (6′) 100 75 ± 25^(a) 12,5,12 (7′) 75 ± 25^(a) 75 ± 25^(a) PQ-11,11 (8′) 50 50 12,2,0,2,12 (9′) 75 ± 25^(a) 100 12,2,1,2,12 (10′) 50 25 12,3,0,3,12 (11′) 100 50 12,3,1,3,12 (12′) 100 50 ^(a)MBEC values varied between 50 and 100 μM for the six trials.

The minimum biofilm eradication concentration (MBEC) of other compounds are shown in Table 2. The inventors have determined that both cationic character and alkyl side chains are important for biofilm eradication. Triscationic compounds (12,2,1,2,12) and (12,3,1,3,12) also showed good capability for film eradication. Compound (12,3,1,3,12) and biscationic compound (12,3,0,3,12) showed similar results. There is a moderate increase in efficacy between bis-cationic (12,2,0,2,12) and tris-cationic (12,2,1,2,12) that provides the most active biofilm-eradicating compound reported among these compound. To demonstrate the activity of these compounds, crystal violet staining and confocal imaging was utilized (FIG. 3). Crystal violet staining illustrates the significant effect that QACs (12,3,12), (12,3,0,3,12), and (12,3,1,3,12) display against pre-established biofilms when compared to the aqueous control or norspermidine. Furthermore, confocal microscopy reveals significant biofilm perturbation at concentrations as low as 12 μM, significantly lower than the reported MBEC values.

In summary, the inventors have demonstrated for the first time that QACs, are potent eradicators of pre-established bacterial biofilms. This development also provides insight toward the minimal structural features needed to eradicate S. aureus and E. faecalis biofilms; merely two permanent cationic charges with alkyl side chains can confer biofilm disruption capability. The compounds are synthesized in one or two steps from commercially available material, making them attractive alternatives to existing methods for biofilm eradication. Triscationic amphiphiles including (12,2,1,2,12) and (12,3,1,3,12) show remarkable antibiofilm activity.

2. Additional Triscationic and Tetracationic Amphiphiles and their Antimicrobial Activities

Despite numerous reports of the antimicrobial activity of biscationic amphiphiles, there are no investigations to the inventors' knowledge correlating antimicrobial activity to amphiphiles with three or four quaternary ammonium groups. This stands in stark contrast to the wide variety of bioactive natural products (and derivatives thereof) incorporating multiple primary, secondary, and tertiary amines; common examples include spermine, spermidine, squalamine, and others. While these are generally drawn as neutral compounds, they will have multiple cations at physiological pH; related compounds have been investigated as possible biofilm disruptors.

With the ready availability of compounds with three or four tertiary amines at modest commercial cost, the inventors continued correlating bioactivity to amphiphilic structure, specifically the number of cations present, as well as the number and lengths of non-polar side chains. The target set of compounds is summarized in Scheme 3. For the trisamine-inspired structures, compounds were designed with either a five- or seven-atom linker between two quaternary ammonium species (Scheme 3, left). This allows for the incorporation of an additional quaternary ammonium moiety in the center of the structure, which can be attached to either short or long chain alkyl residues. The inventors also aimed to prepare structures based on a tetraamine platform, bearing up to four ammonium residues (Scheme 3, right). The inventors aimed to develop unified synthetic methods for the amphiphiles presented.

The inventors first set out to assemble a set of structures that were strictly biscationic, using the simple n,5,n structures, (Z═CH₂, Scheme 3), where n is the length of alkyl chains, and five is the linker length between the nitrogen atoms. Scheme 4 illustrates the preparation of biscationic amphiphiles of the n,5,n series. Syntheses, as illustrated in Scheme 4, were accomplished by the exposure of 1,5-dibromopentane to a range of dimethyl alkyl amines, providing compounds 1-4 (described below in this section). Yields ranged from 72 to 90%. The bis-alkylated products were recrystallized as necessary to remove any residual starting material or alkyl bromide and provide compounds in high purity.

Scheme 5 illustrates preparation of amphiphiles of the (n,2,0,2,n) series. In preparation for a series of compounds bearing either two or three cationic species, the inventors began with the inexpensive pentamethyl bis(ethylenediamine) as shown in Scheme 5. Alkylation thereof was found to be both selective and high-yielding, providing five structures of the (n,2,0,2,n) series, where n is the length of alkyl chains, 2 is the linker length between the nitrogens, and 0 represents the lack of additional substitution on the central nitrogen.

Synthesis of an exemplary compound (14,2,0,2,14) is described herein as representative procedure of preparing compound (m, 2,0, 2,n). To a solution of 1-bromotetradecane (2.25 mL, 8.26 mmol) in CH₃CN (2 mL) was added N,N,N′,N″,N″-pentamethyldiethylenetriamine (0.87 mL, 4.2 mmol). The resulting clear solution was stirred at rt for 20 h, during which time a white solid was observed. To the reaction mixture was added cold acetone (˜9 mL), which led to a white precipitate. Filtration through a Buchner funnel furnished a white solid, which was washed with cold acetone (˜4 mL) and then hexanes (˜4 mL), affording (14,2,0,2,14) (2.188 g, 73%) as a white solid.

Further functionalization of compounds of the n,2,0,2,n series was not as straightforward as the inventors had hoped, as attempted alkylation with long-chained electrophiles (e.g., dodecyl bromide) proved sluggish. However, the inventors were able to effect alkylations (Scheme 6) under stronger conditions, either in neat iodomethane at room temperature (7d, providing n,2,1,2,n) or in allyl bromide at reflux (overnight, providing n,2,3A,2,n). Methylation yields were high 90%), and allylation yields were good (46-83%). Scheme 6 illustrates the preparation of amphiphiles of the type (n,2,1,2,n) and (n,2,3A,2,n).

Synthesis of an exemplary compound (12,2,1,2,12) is described herein as representative procedure of preparing compound (m, 2,1, 2, n). To a solution of CH₃I (˜1.0 mL, 16 mmol) was added (12,2,0,2,12) (201 mg, 0.299 mmol). The resulting clear yellow solution was stirred at room, and additional CH₃I was added over 72 hours, during which time a solid was observed. Crude ¹H NMR showed that (12,2,1,2,12) was the major product.

Scheme 7 shows the preparation of amphiphiles with 7-atom linkers of the type (12,7,12) and (12,3,x,3,12). Suspecting that the proximity of the three cationic nitrogens was an impediment to this final alkylation, the inventors prepared an analogous set of compounds with seven-atom linkers between the “bookend” quaternary ammonium species, maintaining the dodecyl side chains. This began with the preparation of (12,7,12) (Scheme 7) in accordance with the method above. Then, starting with known amphiphile (12,3,0,3,12) (derived from commercially available 2,6,10-trimethyl-2,6,10-triazaundecane) we were able to prepare a set of structures with varied substitution at the center carbon. The inventors first prepared substitutions of methyl and allyl (12,3,1,3,12) and (12,3,3A,3,12), respectively, to parallel the above compounds (12,2,1,2,12) and (12,2,3A,2,12). Simple alkylation was accomplished in good yield with both butyl and dodecyl chains (12,3,4,3,12) and (12,3,12,3,12), respectively, from reflux with the corresponding alkyl bromide. Additionally, both benzyl and para-octyl benzyl groups were prepared as well.

Scheme 8 illustrates the preparation of amphiphiles of the type (N4-n,n,n,n). To further extend the scope of the investigation, the inventors prepared a series of compounds bearing four amines, starting with the commercially available 1,1,4,7,10,10-hexamethyltriethylenetetramine (abbreviated as N4-0,0,0,0), shown in Scheme 8. This opened the possibility of evaluating amphiphiles with up to four cations. The inventors found that monoalkylation of (N4-0,0,0,0) was somewhat selective under our standard conditions (1 equiv RBr, acetonitrile, Δ), providing (N4-12,0,0,0) and (N4-14,0,0,0) in 53-65% yield after trituration. Subsequent alkylation of 29 with 1 equivalent of dodecyl bromide led to a clean preparation of the pseudosymmetric (N4-14,0,0,12), wherein solely the peripheral nitrogens were alkylated, as evidenced by NMR spectra that displayed high levels of symmetry. Alternatively, bis-alkylation of the starting tetraamine furnished either (N4-12,0,0,12) or (N4-14,0,0,14) (93 and 84%, respectively). Bis-ammonium compound N4-12,0,0,12 was in turn bis-allylated under forcing conditions (neat allyl bromide, reflux, 2.5 h) to provide a tetracationic derivative (N4-12,3A,3A,12) (91%).

With nearly three-dozen amphiphiles varying in structure and cationic nature synthesized, minimum inhibitory concentration (MIC) values against the Gram-positive Staphylococcus aureus and Enterococcus faecalis and the Gram-negative Escherichia coli and Pseudomonas aeruginosa were determined by standard methods. Table 3 shows the MIC values for prepared amphiphilic compounds, reported in μM. The commercially available antibacterial agent benzalkonium chloride (benzyldimethyldodecylammonium chloride, Bn,12) was tested for comparison. The broth microdilution for determining MIC values was performed as previously reported.

The MIC data obtained for the 32 amphiphiles tested were found to be remarkably consistent, with over half of compounds tested (20/32) displaying single digit MICs versus all four bacteria tested. MIC variation mostly hinged on the length of alkyl chain, where the dodecyl chain was generally found to be optimal. This was exemplified in the comparison of the X═CH₂ compounds, in both the n,5,n series (1-4) as well as (12,7,12) (20), to the corresponding structures bearing the N—CH₃ group (5, 7-9, 21) or quaternary ammonium groups (10, 12-13, 22, etc) in the center of the compound; little was changed across the board. In fact, (12,5,12) and (12,7,12) were two of the most potent compounds we prepared, with MIC values ≦2 μM in 7 of the 8 cases tested. Compound (12,2,1,2,12) was found to be sparingly soluble in water, and thus posed difficulty in providing repeatable MIC values. In light of the result that longer linkers provided comparable activity, the inventors evaluated the greater structural variety available in the (12,3,x,3,12) series. Finally, the tetraamine scaffold allowed for comparison of compounds with 1, 2, or 4 ammonium groups (e.g., N4-12,0,0,0, N4-12,0,0,12, and N4-12,3A,3A,12). While (N4-12,3A,3A,12) did indeed show strong activity (MIC values=1 μM versus three bacteria, and 2 μM versus P. aeruginosa), N4-12,0,0,12 was equally active, excepting a value of 4 μM versus P. aeruginosa. These both were about 8-fold more bioactive than (N4-12,0,0,0), which bears just one quaternary ammonium group.

In summary, polycationic amphiphiles provided in this disclosure provide great ability to disrupt persistent biofilms and display some level of preferential activity at the more anionic membrane of prokaryotes.

TABLE 3 No. Compound Charge (# nitrogens) S. aureus E. faecalis E. coli P. aeruginosa 1 10, 5, 10 2+ (2N) 2 4 8 63 2 12, 5, 12 2+ (2N) 2 1 1 2 3 14, 5, 14 2+ (2N) 1 1 4 16 4 16, 5, 16 2+ (2N) 2 2 16 32 5 10, 2, 0, 2, 10 2+ (3N) 2 8 8 63 6 11, 2, 0, 2, 11 2+ (3N) 2 2 2 8 7 12, 2, 0, 2, 12 2+ (3N) 1 1 2 4 8 14, 2, 0, 2, 14 2+ (3N) 2 1 4 16 9 16, 2, 0, 2, 16 2+ (3N) 4 2 16 32 10 10, 2, 1, 2, 10 3+ (3N) 2 8 8 63 11 11, 2, 1, 2, 11 3+ (3N) 2 2 4 8 12 12, 2, 1, 2, 12 3+ (3N) ≦0.25-4^(a) ≦0.25-4^(a) 0.5-4^(a) 1-8^(a) 13 14, 2, 1, 2, 14 3+ (3N) 1 1 2 8 14 16, 2, 1, 2, 16 3+ (3N) NT NT NT NT 15 10, 2, 3A, 2, 10 3+ (3N) 2 4 2 63 16 11, 2, 3A, 2, 11 3+ (3N) 1 2 2 16 17 12, 2, 3A, 2, 12 3+ (3N) 2 2 2 4 18 14, 2, 3A, 2, 14 3+ (3N) 2 2 2 8 19 16, 2, 3A, 2, 16 3+ (3N) 4 4 8 16 20 12, 7, 12 2+ (2N) 1 1 2 8 21 12, 3, 0, 3, 12 2+ (3N) 1 2 1 4 22 12, 3, 1, 3, 12 3+ (3N) 1 2 2 8 23 12, 3, 3A, 3, 12 3+ (3N) 1 2 2 4 24 12, 3, 4, 3, 12 3+ (3N) 1 1 1 2 25 12, 3, Bn, 3, 12 3+ (3N) 1 1 1 2 26 12, 3, Bn-8, 3, 12 3+ (3N) 1 1 1 4 27 12, 3, 12, 3, 12 3+ (3N) 0.5 1 1 4 28 N4-12, 0, 0, 0 1+ (4N) 8 8 8 32 29 N4-14, 0, 0, 0 1+ (4N) 16 16 32 125 30 N4-14, 0, 0, 12 2+ (4N) 1 1 2 8 31 N4-12, 0, 0, 12 2+ (4N) 1 1 1 4 32 N4-14, 0, 0, 14 2+ (4N) 2 1 2 16 33 N4-12, 3A, 3A, 12 4+ (4N) 1 1 1 2 34 N4-12, 1, 1, 12 4+ (4N) NT NT NT NT — Bn, 12 1+ (1N) 8 8 32 63 ^(a)Compound (12, 2, 1, 2, 12) showed modest water solubility, and thus provided varying MIC values.

3. Exemplary Compound (12,3,2,3,12)

An exemplary compound (12,3,2,3,12) was prepared through a two-step process.

Step 1: Preparation of Compound (12,3,0,3,12)

To a solution of 2,6,10-trimethyl-2,6,10-triazaundecane (2.77 mL, 2.30 g, 11.4 mmol) in acetonitrile (2.0 mL) was added 1-bromododecane (5.45 mL, 5.66 g, 22.7 mmol). The resulting colorless solution was heated to reflux for 48 h, during which time a golden paste was observed. After cooling, hexane (˜15 mL) was added to the reaction mixture. Filtration though a Buchner funnel furnished a yellow-gold gel, which was washed with cold hexanes (˜5 mL) to afford compound (12,3,0,3,12) (7.95 g, ˜100%) as a yellow-gold gel.

Step 2: Preparation of Compound (12,3,2,3,12).

To the semi-solid trisamine (12,3,0,3,12) (572.3 mg, 0.818 mmol) was added bromoethane (2 mL, 26 mmol). The resulting pale yellow-orange solution was warmed to reflux with stirring for 22 h, during which time a white precipitate was observed. After addition of cold acetone (10 mL) to the still-warm reaction flask, the precipitate was filtered through a Büchner funnel, rinsing with cold acetone (3 mL), before being dried in vacuum, affording 12,3,2,3,12 as a white powder (0.5438 g, 82%), as a white solid; mp=217-219° C.; ¹H NMR (300 MHz, CDCl₃) δ 3.91 (s, 4H), 3.79 (s, 8H), 3.47-3.36 (m, 17H), 2.58 (s, 4H), 1.77 (s, 4H), 1.50 (t, 3H), 1.33-1.16 (m, 36H), 0.86 (t, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 66.39, 61.18, 57.53, 51.04, 49.27, 31.86, 29.59, 29.51, 29.47, 29.28, 26.37, 22.96, 22.62, 18.32, 14.04, 9.02.

Compound (12,3,2,3,12) has showed excellent antimicrobial activities. For example, the values of the MIC (in μM) and the MBEC for each bacterium evaluated are as follows:

Bacteria MICs (μM): S. aureus (SH1000) 0.5 E. faecalis (OG1RF) 0.5 E. coli (MC4100) 1 P. aeruginosa (PA01) 2 CA-MRSA (USA300-0114) 0.5 HA-MRSA (ATCC33591) 1 MBECs (μM): S. aureus 100 E. faecalis 100 USA300-0114 100 Lysis20: 8

No resistance was observed in liquid cultures of SH1000 or USA300-0114 serially passaged with Compound (12,3,2,3,12) over a period of at least 14 days.

4. Exemplary Compound (12,3,11-SH,3,12)

An exemplary compound (12,3,11-SH,3,12) having the following formula was prepared as follows.

To a solution of 11-bromo-1-undecanethiol (0.1250 g, 0.4677 mmol) in acetonitrile (˜1 mL) was added a trisamine compound (12,3,0,3,12) (0.3273 g, 0.4538 mmol). The resulting colorless solution was heated at reflux under argon for 1 week. Addition acetonitrile (1 mL) was added to the reaction flask after 2 days and 3 days to facilitate dissolution. To the warm reaction mixture was added cold hexanes (˜6 mL), during which time a yellowish precipitate was observed. Filtration through a Buchner funnel furnished a yellowish amorphous solid, which was washed with cold hexanes (˜10 mL) to afford 12,3,11-SH,3,12 (0.170 g, 80%) as a yellow amorphous solid; ¹H NMR (300 MHz, CDCl₃) δ 3.73 (s, 8H), 3.42 (s, 6H), 3.33-3.27 (m, 15H), 3.09 (s, 1H) 2.45-2.40 (m, 6H), 1.72 (s, 6H), 1.56-1.48 (m, 6H), 1.29-1.19 (m, 46H), 0.81 (t, J=6.5 Hz, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 66.19, 66.02, 64.21, 61.55, 60.92, 58.07, 51.73, 51.18, 50.95, 50.02, 41.56, 33.94, 32.15, 31.83, 29.66, 29.57, 29.27, 28.96, 28.85, 28.27, 26.32, 24.55, 22.88, 22.59, 18.67, 18.29, 14.01; high resolution mass spectrum (ESI) m/z 242.2514 ([M]³⁺; calculated for [C₄₆H₁₀₀N₃S]³⁺: 242.2541).

This compound (12,3,11-SH,3,12) has showed excellent antimicrobial activities. For example, the values of the MIC (in μM) and the MBEC for each bacterium evaluated are as follows:

Bacteria MICs (μM): S. aureus 2 E. faecalis 2 E. coli 2 P. aeruginosa 8 USA300-0114 2 ATCC 33591 2 MBECs (μM): S. aureus 200 E. faecalis >200 USA300-0114 >200

5. Additional Variations and Examples:

A compound having the formula (I) or (II) is described above. In some embodiments, all the nitrogen atoms are ionized. In some other embodiments, ionization of some nitrogen atoms such as the middle nitrogen(s) are optional. For example, ionization of the middle nitrogen (N) atom due to substitution of either R₃ or R₄ in Formula (I), as well as the according counterion Y may be optional. In other words, a compound having formula (I) has no Y anion and only has one substituent group (either R₃ or R₄). Alternatively, either R₃ or R₄ can be considered as a lone pair of electrons and the central N would be neutral. Similarly, ionization of the two middle nitrogen (N) atoms due to substitution of either R₃ or R₄ and either R₅ or R₆ in Formula (II), as well as the according counterions Y⁻, may be also optional. For example, the compounds having formula (I) or (II) could have the formula

In some embodiments, these compounds such as the compound having formula I-6 or 11-6 can become ionized at the central nitrogen in a solution, possibly due to the effect of pH.

In some preferred embodiments, the compound has the formula

as described before or

In the preferred embodiments, each of m and n is 10, 11 or 12, and each of R₁, R₂, R₃, R₄, R₅ and R₆ is methyl or ethyl. R₄ may be also hydrogen.

As described above, some exemplary compounds are denoted using a combination of numbers. For example, in some compounds having the formula (I-2), (I-3), (I-4) (I-5), (I-6) or (I-7), both R₁ and R₂, both R₅ and R₆ are methyl. At least one of R₃ and R₄ is methyl. Assuming R₃ is methyl, R₄ is optionally H or an alkyl having x carbons, which is substituted or optionally substituted. Such a compound is denoted as compound [m, s, x, s, n], compound (m, s, x, s, n), or compound m, s, x, s, n. s is the number of carbon atoms between two nitrogen atoms. x is the number of carbon atoms in R₄. When R₄ is H, x is zero. To distinguish from an ionized compound having Formula (I-3) with R₄ being H in formula (I-3) in which x is labelled as 0′, x is labelled as 0 for a compound having Formula (I-7) without R₄ substitution.

For example, one preferred compound is compound (10, 3, 0, 3, 10), which has the formula:

The middle nitrogen (N) atom in the compound (10, 3, 0, 3, 10) can be further substituted and ionized through synthesis or during use in a solution such as an aqueous solution. For example, one exemplary compound is compound (10, 3, 0′, 3, 10) having the formula:

or compound (10,3,1,3,10) having the formula:

The exemplary compound (10,3,0,3,10) having the following formula (which is the same as described above) was prepared as follows.

To a solution of 2,6,10-trimethyl-2,6,10-triazaundecane (1.39 mL, 5.73 mmol) in acetonitrile (1 mL) was added 1-bromododecane (2.37 mL, 11.5 mmol). The resulting colorless solution was heated at reflux with stirring for 24 h, during which time a pale-yellow paste was observed. After addition of cold hexanes (˜10 mL) to the reaction flask, the precipitate was filtered through a Büchner funnel, rinsing with cold hexanes (˜5 mL), affording compound (10, 3,0, 3,10) (2.40 g, 99%) as a pale yellow paste; ¹H NMR (300 MHz, CDCl₃) δ 4.00-3.95 (m, 4H), 3.41-3.29 (m, 16H), 2.54-2.50 (m, 4H), 2.18 (s, 3H), 1.88-1.80 (m, 4H), 1.72-1.60 (m, 4H), 1.28-1.19 (m, 28H), 0.81 (t, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 64.21, 63.35, 53.93, 50.95, 41.85, 31.73, 29.33, 29.13, 26.29, 22.81, 22.52, 20.81, 13.95; high resolution mass spectrum (ESI) m/z 241.7747 ([MH]²⁺; calculated for [C₃₁H₆₉N₃]²⁺: 241.7746).

Similarly, other exemplary compounds including compounds (12,2,0,2,12) and (12,3,0,3,12) were prepared. Compounds (12,2,0,2,12) and (12,3,0,3,12) has formula (I-6), in which R₁, R₂, R₃, R₅, and R₆ are methyl.

An exemplary compound (10,3,2,3,10) having the following formula was prepared as follows.

To the semi-solid trisamine (10,3,0,3,10) (305 mg, 0.721 mmol) was added bromoethane (2.0 mL, 27 mmol). The resulting pale yellow transparent solution was warmed to reflux with stirring for 20 h, during which time an opaque white paste was observed, impeding stirring. To the reaction flask was added bromoethane (˜1 mL); this was continued at reflux for an additional 4 h. After addition of cold hexanes (10 mL) to the still-warm reaction flask, the precipitate was filtered through a Buchner funnel, rinsing with cold hexanes (5 mL), affording 10(3)2(3)10 (0.340 g, 89%), as a white amorphous solid; ¹H NMR (300 MHz, CDCl₃) δ 3.91 (s, 4H), 3.79 (s, 8H), 3.48-3.36 (m, 17H), 2.59 (s, 4H), 1.77 (s, 4H), 1.48 (t, 3H), 1.34-1.09 (m, 44H), 0.87 (t, 6H); ¹³C NMR (75 MHz, CDCl₃) δ 66.13, 61.18, 59.29, 57.52, 51.35, 50.98, 49.10, 31.82, 29.48, 29.28, 29.25, 26.37, 22.93, 22.59, 18.07, 14.01, 8.97; high resolution mass spectrum (ESI) m/z 255.7902; ([M-H]²⁺; calculated for [C₃₃H₇₃N₃]²⁺: 255.7902).

Other compounds as shown in FIG. 4 were evaluated with data shown in Tables 4 and 5.

For example, Compound PQ-12, Bn having the following formula was prepared as follows.

To a solution of benzylbromide (0.100 mL, 0.846 mmol) in acetonitrile (1 mL) was added PQ-12,0 (0.172 g, 0.423 mmol). The colorless solution was warmed to reflux with stirring, during which time a yellow solid precipitated, impeding stirring. The reaction continued at reflux for 24 h. After addition of cold acetone (˜2 mL) to the reaction flask, the precipitate was filtered through a Buchner funnel, rinsing with cold acetone (˜6 mL). The solid was further washed with warm hexanes (˜4 mL), filtered through a Buchner funnel, rinsing with cold hexanes (˜10 mL) and allowed to dry affording PQ-12,Bn (0.1674 g, 69%) as a yellow solid; mp=273-275° C.; ¹H NMR (300 MHz, CD3OD) δ 9.31 (dd, J=6.5 Hz, 4H), 8.69-8.65 (m, 4H), 7.59-7.57 (m, 2H), 7.51-7.49 (m, 3H), 5.97 (s, 2H), 4.73 (t, J=7.7 Hz, 2H), 2.13-2.04 (m, 2H), 1.41-1.28 (m, 18H), 0.89 (t, J=6.8, 3H); 13C NMR (75 MHz, CD3OD) δ 150.36, 145.64, 129.84, 129.39, 129.01, 127.18, 127.00, 64.54, 61.95, 31.60, 31.11, 29.27, 29.18, 29.05, 28.99, 28.70, 25.82, high resolution mass spectrum (ESI) m/z 208.1594 ([M−H]²⁺; calculated for [C₂₉H₄₀N₂]²⁺: 208.1596). 22.26, 12.96).

Different biological assays were performed. For all biological assays, laboratory strains of methicillin-susceptible Staphylococcus aureus MSSA (SH1000), Enterococcus faecalis (OG1RF), Escherichia coli (MC4100), Pseudomonas aeruginosa (PAO1), community-acquired methicillin-resistant Staphylococcus aureus CA-MRSA (USA300-0114), and hospital-acquired methicillin-resistant Staphylococcus aureus HA-MRSA (ATCC 33591) were grown at 37° C. overnight from freezer stocks in 10 mL of the media indicated for each assay. All cultures, with the exception of E. faecalis, were grown with shaking at 250 rpm.

Minimum inhibitory concentration (MIC) values against bacteria were measured using standard method. Compounds were serially diluted two-fold from an aqueous stock solution to yield twelve test concentrations. Overnight S. aureus, E. faecalis, E. coli, P. aeruginosa, CA-MRSA, and HA-MRSA cultures diluted to ca. 10⁶ cfu/mL in Mueller-Hinton media and 100 μL were inoculated into each well of a U-bottom 96-well plate (BD Biosciences, BD351177) containing 100 μL of compound solution. Plates were incubated statically at 37° C. for 72 hours upon which time wells were evaluated visually for bacterial growth. The MIC was determined as the lowest concentration of compound resulting in no bacterial growth visible to the naked eye, based on the majority of three independent experiments. Positive and negative aqueous and media controls were conducted for each trial.

Minimum biofilm eradication concentration (MBEC) was also measured. To each well of a flat-bottomed 96-well plate (Corning 3370), 8 μL of overnight bacterial culture was brought to a volume of 200 μL with fresh media (BHI for S. aureus and MRSA, Todd Hewitt (TH) for E. faecalis). Plates were incubated statically for 24 hours at 37° C. to establish biofilms. After 24 hours, the wells were carefully emptied by inverting the plate and gently shaking. A pre-mixed solution of media and aqueous compound stock solution was added to each well and plates were incubated at 37° C. 16 hours after pre-established biofilms were treated with compound, the media from each well was removed, biofilms were washed three times with 200 μL PBS to remove planktonic cells, and biofilms were incubated overnight at 37° C. in 200 μL of fresh media to allow any viable cells to continue growing. The wells were then vigorously pipetted to resuspend the biofilm cells and the OD at 595 nm was measured using a plate reader (POLARstar Omega, BMG Labtech). Biofilms reaching a final OD of less than 0.1 were considered eradicated and the lowest concentration of antibiotic corresponded to the MBEC. Four replicates were completed for each concentration of compound as well as positive and negative controls.

Hemolysis assays (Lysis₂₀) were performed according to the procedure detailed in Peng, L., DeSousa, J., Su, Z., Novak, B. M., Nevzorov, A. A., Garland, E. R., and Melander, C., Inhibition of Acinetobacter baumannii biofilm formation on a methacrylate polymer containing a 2-aminoimidazole subunit, Chem. Comm. 2011, 47, 4896-4898. Defibrinated sheep blood was purchased from Hemostat Labs (DSB 100). Compounds representative of each class were serially diluted in sterile PBS from aqueous stock solutions and incubated with resuspended blood cells. The OD of the final suspensions were measured directly using plate reader. TritonX (1% by volume) served as a positive control (100% lysis marker) and sterile PBS served as a negative control (0% lysis marker).

Resistance development such as liquid culture resistance assay was performed. Single colonies of MSSA (SH1000) and MRSA (USA300-0114) were inoculated into TH media overnight. Each culture was then inoculated into vials of TH media containing two-fold dilutions of antibacterial compound in the range of the MIC 0.25×MIC, 0.5×MIC, MIC, and 2×MIC) and incubated with shaking at 37° overnight. Once a day, an aliquot of bacterial culture from the highest concentration allowing bacterial growth was diluted 1:10,000 in fresh media containing a range of antibacterial compound. This process was repeated for several hundred generations.

Table 4 shows minimum inhibitory concentration (MIC) for QACs against Gram-positive E. faecalis, and Gram-negative strains E. coli and P. aeruginosa. Lysis₂₀ indicates the concentration of compound (in μM) at which less than 20% of red blood cells are lysed.

MIC (μM) CA- HA- Compound MSSA MRSA MRSA Lysis₂₀ Bn,12 8 32 8 63 10,10 1 4 2 16 Pyr,16 0.5 16 1 8 12,3,0 4 8 4 32 16,2,0 1 4 2 16 12,2,12 1 0.5 0.5 8 12,3,12 2 1 2 8 12,5,12 2 1 0.5 8 16,5,16 2 8 1 4 12,2,0,2,12 1 1 0.5 8 10,3,0,3,10 1 2 2 32 12,3,0,3,12 1 1 0.5 4 12,2,3A,2,12 2 1 1 2 12,2,1,2,12 1 1 1 16 12,3, 3A, 3,12 1 1 1 8 12,3,1,3,12 1 0.5 1 8 10,3,2,3,10 2 2 2 32 12,3,2,3,12 0.5 0.5 1 8 12,3,4,3,12 1 0.5 0.5 8 12,3,Bn,3,12 2 1 1 8 12,3,Bn-8,3,12 2 2 1 8 12,3,12,3,12 0.5 1 1 4 12,3,11- 2 2 2 16 SH,3,12 N4-12,0,0,12 1 1 1 4 N4- 1 1 0.5 8 12,3A,3A,12 PQ-12,12 1 1 0.5 8 MQ-12,12 1 2 1 8 PMQ-12,12 1 1 1 8 PQ-12,Bn 4 32 16 125 MQ-12,Bn 8 16 16 125

Table 5 shows minimum biofilm eradication concentration (MBEC) for QACs against Gram-positive susceptible and resistant strains. MSSA represents SH1000, CA-MRSA represents USA300-0114.

MBEC (μM) Compound E. faecalis MSSA CA-MRSA Bn,12 ≧200 >200 ≦200 10,10 >200 150 ± 50  ≧200 Pyr,16 50 50 200 12,3,0 >200 >200 >200 16,2,0 50 100 >200 12,2,12 50 75 ± 25 200 12,3,12 50 75 ± 25 100 12,5,12 100 50 100 16,5,16 ≧200 200 >200 12,2,0,2,12 100 75 ± 25 100 10,3,0,3,10 50 150 ± 50  150 ± 50 12,3,0,3,12 100 50 150 ± 50 12,2,3A,2,12 75 ± 25 50 100 12,2,1,2,12 >200 200 ≧200 12,3,3A,3,12 75 ± 25 50 100 12,3,1,3,12 200 100 150 ± 50 10,3,2,3,10 100 200 150 ± 50 12,3,2,3,12 100 100 100 12,3,4,3,12 50 75 ± 25  75 ± 25 12,3,Bn,3,12 100 100 200 12,3,Bn-8,3,12 200 200 >200 12,3,12,3,12 200 100 >200 12,3,11-SH,3,12 >200 200 >200 N4-12,0,0,12 75 ± 25 50 100 N4-12,3A,3A,12 200 100 150 ± 50 PQ-12,12 200 200 100 MQ-12,12 100 100 100 PMQ-12,12 100 100 150 ± 50 PQ-12,Bn 200 200 200 MQ-12,Bn >200 >200 >200

The main mechanism of QAC resistance in Gram-positive bacteria is the qacA/qacR system, wherein QacR is a negative transcriptional regulator of QacA, a membrane-affiliated efflux pump of the major facilitator superfamily. A key player in QAC-resistance is methicillin-resistant Staphylococcus aureus (MRSA), which is notorious for its multidrug resistance and the resultant difficulties in treating MRSA-affiliated infections that are prominent in hospitals and other close-contact settings. MRSA strains are typically identified as community-acquired (CA-MRSA) or hospital-acquired (HA-MRSA), depending on the location from which they are isolated and the type of chromosomal resistance cassette they contain. Specifically, CA-MRSA strain USA300 has been shown to possess qacA/qacR among the plethora of multidrug resistance systems that this strain carries. The rate at which bacteria have evolved resistance mechanisms to QACs is quite alarming. Interestingly, hospital workers carried a significantly higher portion of QAC-resistant strains within their microbiome as compared to the general population. The spread of resistance may also be exacerbated by the prevalence of biofilms, which are communities of bacteria adhered to a surface and to each other through an extracellular polymeric substance consisting of polysaccharides, proteins, and extracellular DNA. Increased plasmid transfer, hypermutation, and reduced metabolism of bacteria in the biofilm state all contribute towards resistance and render many traditional targets of antibiotics ineffective.

The inventors investigated structure-activity relationships of mono- and multiQACs, focusing on four characteristics: 1) biofilm eradication properties against MRSA and MSSA, 2) antimicrobial activity against resistant bacteria, 3) toxicity to eukaryotic cells, and 4) susceptibility to resistance development in MSSA. A library of compound as shown in FIG. 4 was investigated. These compounds include bis-, tris-, and tetracationic structures with varying alkyl chains and charge distribution. Three commercial monocationic QACs found in standard disinfectants were included as benchmarks due to their prevalence in society (FIG. 4A). The compounds also include monocationic species (FIG. 4B), a series of structurally varied polyamines (FIG. 4C), one tetracationic species (FIG. 4D), compounds from a trisamine scaffold (FIG. 1E), and bis-aromatic structures (FIG. 4F). This collection of QACs was tested against laboratory strains of QAC-susceptible E. faecalis (OG1RF) and S. aureus (SH1000), CA-MRSA (USA300-0114) and HA-MRSA (ATCC 33591), and Gram-negative planktonic bacteria, in addition to MSSA, CA-MRSA, and E. faecalis biofilms (as shown in Tables 4 and 5).

MSSA and MRSA Biofilm Eradication Assays: It has been reported that MRSA biofilms are more robust and difficult to eradicate when compared to MSSA. In accordance with this observation, the inventors found that most commercial monoQACs were 2-4 fold less effective against MRSA biofilms (Table 5). In particular, Pyr,16, which is one of the most effective commercial disinfectants, experiences a four-fold decrease in efficacy against MRSA in the biofilm state. In contrast, over a third of the multiQACs presented in Tables 4 and 5 (9/25) are equipotent against MRSA and remain among the most potent Gram-positive antibiofilm agents reported to date. Taken in combination, these results hint at a multicationic-specific interaction with the biofilm matrix resulting in advantageous properties when compared to commercial monoQACs.

Antibacterial and Hemolytic Studies: Results for antibacterial activity (minimum inhibitory concentration, MIC) are summarized in Table 4. The effect of the number of cations in the QAC structure ranging from two to four was investigated. In some embodiments, installation of a third quaternary ammonium group, the result of alkylation of the central nitrogen of compounds, did not noticeably improve the MIC. For example, compounds (12, 3, 0, 3,12), (12, 3, 1, 3, 12) and (12, 3, 12, 3, 12) (FIG. 4E) demonstrated nearly identical activity despite possessing different numbers of cations (two vs. three) and markedly different substituents at the center carbon (either 1- or 12-carbon chains). Similar results were obtained when comparing tetracationic species (N4-12,3A,3A,12), which displays equipotent activity compared to analogous compounds of lower charge with (N4-12,0,0,12), which contains two tertiary amines. These results indicate that permanent charge does not appear to play a direct role in antibacterial activity.

Given the known toxicity issues of such broad-spectrum antibacterials, the inventors sought to investigate the role substituents played with the goal of identifying a compound with improved antibacterial and hemolytic properties. The inventors first found that twenty to twenty-four side chain carbons provide optimal MIC values were once again observed in this application of QACs in some embodiments. This is highlighted in the comparison of compound (16,5,16) to their counterparts (12,2, 12) and (12, 5,12) (FIG. 4C), with the latter pair displaying a superior antibacterial and hemolytic profile (Table 4). The inventors further evaluated compounds bearing a functionalized side chain for potential surface attachment, such as the allyl and thiol compounds (12,3, 3A, 3,12) and (12, 3,11-SH, 3,12). In the (12, 3, R, 3, 12) series, (12, 3,11-SH, 3, 12) was divergent exhibiting slightly higher MICs and an improved Lysis₂₀ value. The inclusion of benzyl or decyl groups helps mitigate eukaryotic toxicity in QACs, as demonstrated by Bn,12 and 10,10. Taking inspiration from these compounds, The inventors synthesized (10,3,0,3,10), (10,3,2,3,10), MQ-12,Bn, and PQ-12,Bn and evaluated their lytic profiles. All four compounds displayed the highest prokaryotic selectivity of the multiQACs evaluated. Taken in sum, derivatization of the alkyl sidechains provides an opportunity to tune both the antibacterial activity and cell toxicity providing compounds with improved profiles when compared to commercial mainstays as evidenced by (10,3,0,3,10).

BisQAC PQ-12,Bn displayed elevated MICs against both MRSA strains, despite a lack of previous exposure in the environment. A structural comparison of PQ-12,Bn to other bioactive QACs presented suggests that the high degree of conjugation is responsible for this response. This is to our knowledge the first case of the qacA/qacR system presumably recognizing and effluxing a novel QAC substrate to which the bacteria has not previously been subjected. This finding, in combination with the multiple crystal structures containing aromatic dyes, lends credence to the notion that aromatic interactions in the active site of QacR, the negative transcriptional regulator of QacA expression, are important in QacR binding and activation. Alternatively, one could postulate that the aryl moieties improve cell permeability; therefore, more readily interacting with QacR and triggering production of QacA. This would be analogous to the high propensity of aryl residues in cell-penetrating peptides which have been shown to increase their cell permeability.

Resistance Development Assays: Liquid culture serial passage protocols, in which bacteria are successively grown at sub-lethal concentrations of antibacterial compound, were employed. Cultures of MSSA (SH1000) passaged with sub-lethal doses of Bn,12 for 31 days (>700 generations) experienced consistent growth when moved to an elevated concentration (2×MIC at day 11; 4×MIC at day 24), indicating the development of resistance to the monocationic, aryl-containing QAC. Similar results were obtained for MSSA grown in the presence of commercially produced 10,10 (10 days, 240 generations). No resistance was observed to the broadly active biscationic (12,3,12), triscationic (12,3,2,3,12), and tetracationic (12,2,3A,2, 3A, 2,12) against MSSA and against a CA-MRSA strain that carries QAC-resistance genes (USA300-0114) over 24 days (>500 generations), hinting that a novel, and not an iterative, resistance mechanism is necessary for bacterial survival. The resistance mechanism to monoQACs involves just a single amino acid change to recognize the cationic nitrogen. Resistance to mono- and bisQACs bearing aryl substituents might be more readily acquired when compared to multicationic structures.

MultiQACs are potent disinfectants and biofilm eradicators against MSSA and MRSA including strains carrying QAC-resistance genes. QAC compounds bearing the aromatic benzyl moiety such as commercial Bn,12, Pyr,16, and novel PQ-12,Bn showed decreased potency when tested against a MRSA strain possessing qacA/R, implying that aromatic moieties in addition to their mono- or biscationic nature is key for substrate recognition within the QAC-resistance system. Furthermore, MSSA was able to independently develop resistance to mono- and bisQACs possessing aryl substituents within a few hundred generations, something not observed for multiQACs. While this invention is bound by any theory, the data appear to suggest that in order to combat multiQACs, bacteria must develop a mechanism of QAC-resistance that is different from the qacA/R resistance mechanism.

Although the subject matter has been described in terms of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be construed broadly, to include other variants and embodiments, which may be made by those skilled in the art. 

What is claimed is:
 1. An antimicrobial composition comprising a compound having the formula

wherein: R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is H or a C₁₋₁₂ alkyl unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —N—C(O)R′, —N—C(O)CR′═CR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, —OCF₃, halogen, benzyl, o-vinylbenzyl, m-vinylbenzyl, p-vinylbenzyl, phenyl, allyl, and substituted allyl; R₇, R₈ or R₉ is a C₁₋₁₂ alkyl unsubstituted or optionally substituted with a functional group selected from the group consisting of —OH, —OR′, —NH₂, —NHR′, —SH, —SR′, —O—C(O)R′, —C(O)R′, —CF₃, and —OCF₃, R′ is H or a C₁₋₄ alkyl; X or Y is a halogen; and m and n are integers in the range from 1 to 25; wherein ionization of the middle nitrogen (N) atom due to substitution of either R₃ or R₄ in Formula (I), as well as the according counterion Y−, or ionization of the two middle nitrogen (N) atoms due to substitution of either R₃ or R₄ and either R5 or R6 in Formula (II), as well as the according counterions Y−, are optional.
 2. The antimicrobial composition of claim 1, wherein the compound has the formula


3. The antimicrobial composition of claim 1, wherein R₁, R₂, R₃, R₄, R₅, R₆, R₁₀ or R₁₁ is H or a C₁₋₄ alkyl; and R₇, R₈ or R₉ is a C₂₋₅ alkyl unsubstituted or optionally substituted.
 4. The antimicrobial composition of claim 1, wherein at least one of R₁, R₂, R₃, R₄, R₅, R₆, R₁₀, or R₁₁ is a C₁₋₁₂ alkyl substituted with a functional group selected from the group consisting of —SH, allyl, and substituted allyl.
 5. The antimicrobial composition of claim 1, wherein X or Y is chlorine or bromine; and m and n are integers in the range from 10 to
 16. 6. The antimicrobial composition of claim 1, wherein the compound has the formula


7. The antimicrobial composition of claim 6, wherein each of m and n is 10, 11 or 12, and each of R₁, R₂, R₃, R₅ and R₆ is methyl or ethyl, and R₄ is hydrogen, methyl or ethyl.
 8. The antimicrobial composition of claim 1, wherein the compound is compound (10, 3, 0, 3, 10) having the formula:


9. The antimicrobial composition of claim 7, wherein the middle nitrogen (N) atom of the compound (10, 3, 0, 3, 10) is further substituted and ionized.
 10. The antimicrobial composition of claim 1, wherein the compound is compound (10, 3, 0′, 3, 10) having the formula:

or compound (10,3,1,3,10) having the formula:


11. A method of making the antimicrobial composition of claim 1 comprising mixing an effective amount of a compound having the formula (I) or (II) and a carrier.
 12. A method of killing or inhibiting microbial growth, comprising applying the antimicrobial composition of claim 1 comprising a compound having the formula (I) or (II).
 13. The method of claim 12, wherein the antimicrobial composition is used to kill or inhibit growth of at least one group of microorganisms selected from the group consisting of bacteria, viruses, yeast, fungi, and protozoa, or to inhibit formation of a biofilm or eradicate a pre-established biofilm.
 14. The method of claim 12, wherein the compound has the formula


15. The method of claim 14, wherein each of m and n is 10, 11 or 12, and each of R₁, R₂, R₃, R₅ and R₆ is methyl or ethyl, and R₄ is hydrogen, methyl or ethyl.
 16. The method of claim 11, wherein the compound is compound (10, 3, 0, 3, 10) having the formula:


17. The method of claim 16, wherein the middle nitrogen (N) atom of the compound (10, 3, 0, 3, 10) is further substituted and ionized.
 18. The method of claim 12, wherein the compound is compound (10, 3, 0′, 3, 10) having the formula:

or compound (10,3,1,3,10) having the formula: 